1. Field of the Invention
The present invention relates to laser tweezers and Raman spectroscopy (LTRS) systems and methods.
2. Description of the Related Art
Detection and identification of microscopic particles using micro-Raman spectroscopy has been used in various scientific disciplines, including materials science, biology and medicine. G. Turrell and J. Corset, Raman microscopy, (Academic Press, London, 1996). J. H. Laserna, Modern techniques in Raman spectroscopy, (John Wiley & Sons, Chichester, 1996). As an analytical tool, micro Raman spectroscopy uses a focused laser beam to illuminate particles, and the resulting scattered light is detected and analyzed. The incident light on the sample must be administered at an energy level sufficient to excite molecules in the sample to produce inelastic scattering. Even when inelastic scattering occurs, most of the scattered light is elastically scattered, and is called “Rayleigh scatter.” This light is the same wavelength as the incident light. However, some of the light is inelastically scattered due to the excitation of molecules in the sample, resulting in scattered light having a wavelength that is different from the incident light. The inelastically scattered light spectrum is called a Raman spectrum. The molecular composition and structure information of the particles can be obtained from positions, intensities, and line-widths of the Raman peaks in the spectra.
When particles, such as colloid particles and motile biological cells, are dispersed in a liquid, the conventional micro-Raman spectroscopy becomes less effective because the particles will randomly move in and out of the illuminating micro-probe beam due to Brownian motion. In such a case, the particles under study have to be immobilized with either physical or chemical methods.
Optical tweezers are used to trap particles by exploiting the properties of momentum associated with light. When light passes through a fluid medium, the optical path is bent by refraction in the fluid material. The bending of the light path corresponds to a change in momentum of the light. If a dielectric particle is suspended in a fluid media of lower refractive index, the light path bends as shown in FIG. 1. The bending of the light path 11 corresponds to a transfer of momentum from the light to the refracting dielectric particle 13. The transfer of momentum exerts a force, which is capable of holding or manipulating the motion of the particle 13.
Researchers have combined various spectroscopic techniques, including adsorption, fluorescence, and Raman spectroscopy, with optical tweezers to characterize various types of samples. In laser tweezers/Raman spectroscopy (LTRS) systems, the same laser is typically used to both trap a sample particle and to excite molecules in the sample to produce a Raman spectrum. For example, a LTRS system was developed to study single trapped polystyrene beads with a power of 80 mW from a Ti:Sapphire laser. See K. Ajito and K. Torimitsu, Trends Anal. Chem. 20, 255 (2001). The same Ti:Sapphire laser was used for both trapping and excitation.
However, obtaining Raman measurements of small particles may cause severe photodamage, particularly to living cells. Relatively small particle samples confined to a small space by laser tweezers require a high-intensity excitation in order to obtain a Raman measurement. Such high-intensity excitation over time can cause severe photodamage and destroy the sample. For example, photodamage to single Eschericia coli cells in an optical trap were observed after a few minutes of optical trapping with a continuous wave (cw) laser. K. Neuman, E. H. Chadd, G. F. Liou, K. Bergman, and S. M. Block, Biophys. J. 77, 2656 (1999).
On the other hand, Raman spectroscopy of small particle samples such as single living cells could prove useful for providing information about species, structures, and molecular conformation with the particles being studied. Raman spectroscopy may provide a fingerprint for the identification of microscopic particles.
Raman spectroscopy and optical trapping may also be useful in studying particles having a high index of refraction and high coefficients of absorption. Living biological cells and microdroplets are typically relatively transparent and have a low relative index of refraction. Stable trapping with a single Gaussian beam may be obtained with such particles, in part, because any force produced by scattered light is minimal in comparison to the gradient force produced by the change in momentum of the laser through the surrounding fluid. However, for particles having a high index of refraction and high coefficients of reflection and absorption, such as metallic particles, it is more difficult to achieve stable trapping with a single Gaussian beam. The gradient force produced by the laser may be small in comparison with the enlarged scattering force. Such a scattering force generally points in the direction of the incident laser beam and tends to repulse the particles from the laser beam. A. Ashkin, J. M. Dziedzic, J. E. Bjorkholm, and S. Chu, Opt. Lett. 11, 228-290 (1986). A. Ashkin, J. M. Dziedzic and T. Yamane, Nature, 330, 769-771 (1987). K. Svoboda and S. M. Block, Annu. Rev. Biophys. Biomol. Struc. 23, 247-284 (1994).
Several schemes have been developed to confine metal particles and absorptive particles, including techniques using a two-dimensional trapping by a TEMO01-mode laser beam (A. Ashkin and J. M. Dziedzic, Appl. Phys. Lett., 24, 432 (1974). G. Roosen and C. Imbert, Opt. Commun. 26, 432 (1978).), a high-order Laguerre-Gaussian beam (H. He, M. E. J. Friese, N. R. Heckenberg, and H. Rubinsztein-Dunlop, Phys. Rev. Lett. 75, 826-829 (1995). A. T. O'Neil and M. H. Dadgett, Opt. Commun., 185, 139-143 (2000).) and a circularly scanning beam (K. Sasaki, M. Koshioka, H. Misawa, N. Kitamura, and H. Masuhara, Appl. Phys. Lett. 60, 807-809 (1992).). These methods rely on the repulsive scattering force to trap the non-transparent particles in the dark central minimum of a doughnut-shaped beam. Metal particles have also been confined to two-dimensions with a fixed Gaussian beam at an off-axial position based on the attractive force arising from a creeping wave (H. Furukawa and I. Yamaguchi, Opt. Lett. 23, 216-219 (1998)), or at an off-focus position when the laser beam focus is located near the bottom of the particle (Shunichi Sato, Yasunori Harada, and Yoshio Waseda, Opt. Lett. 19, 1807-1809 (1994).). However, these techniques do not propose the combination of Raman spectroscopy with optical trapping for highly refractive and non-transparent particles.
Therefore, there remains a need to develop LTRS systems and methods for optically trapping a sample and subsequently performing Raman spectroscopy on the sample while minimizing photodamage to the sample and/or for studying particles having a high index of refraction and high coefficients of absorption.